Medium supplementation can influence the human ovarian cells in vitro

Background Cells are an essential part of the triple principles of tissue engineering and a crucial component of the engineered ovary as they can induce angiogenesis, synthesize extracellular matrix and influence follicle development. Here, we hypothesize that by changing the medium supplementation, we can obtain different cell populations isolated from the human ovary to use in the engineered ovary. To this end, we have in vitro cultured cells isolated from the menopausal ovarian cortex using different additives: KnockOut serum replacement (KO), fetal bovine serum (FBS), human serum albumin (HSA), and platelet lysate (PL). Results Our results showed that most cells soon after isolation (pre-culture, control) and cells in KO and FBS groups were CD31- CD34- (D0: vs. CD31-CD34+, CD31 + CD34+, and CD31 + CD34- p < 0.0001; KO: vs. CD31-CD34+, CD31 + CD34+, and CD31 + CD34- p < 0.0001; FBS: vs. CD31-CD34+ and CD31 + CD34+ p < 0.001, and vs. CD31 + CD34- p < 0.01). Moreover, a deeper analysis of the CD31-CD34- population demonstrated a significant augmentation (more than 86%) of the CD73+ and CD90+ cells (possibly fibroblasts, mesenchymal stem cells, or pericytes) in KO- and FBS-based media compared to the control (around 16%; p < 0.001). Still, in the CD31-CD34- population, we found a higher proportion (60%) of CD90+ and PDPN+ cells (fibroblast-like cells) compared to the control (around 7%; vs PL and KO p < 0.01 and vs FBS p < 0.001). Additionally, around 70% of cells in KO- and FBS-based media were positive for CD105 and CD146, which may indicate an increase in the number of pericytes in these media compared to a low percentage (4%) in the control group (vs KO and FBS p < 0.001). On the other hand, we remarked a significant decrease of CD31- CD34+ cells after in vitro culture using all different medium additives (HSA vs D0 p < 0.001, PL, KO, and FBS vs D0 P < 0.01). We also observed a significant increase in epithelial cells (CD326+) when the medium was supplemented with KO (vs D0 p < 0.05). Interestingly, HSA and PL showed more lymphatic endothelial cells compared to other groups (CD31 + CD34+: HSA and PL vs KO and FBS p < 0.05; CD31 + CD34 + CD90 + PDPN+: HSA and PL vs D0 p < 0.01). Conclusion Our results demonstrate that medium additives can influence the cell populations, which serve as building blocks for the engineered tissue. Therefore, according to the final application, different media can be used in vitro to favor different cell types, which will be incorporated into a functional matrix.


Background
The ovaries are female reproductive organs that contain four layers, including the germinal epithelium layer, the collagenous connective tissue, the cortex holding preantral follicles, and the medulla comprising loose connective tissue, blood vessels, and antral follicles. Follicles are Page 2 of 12 Dadashzadeh et al. Journal of Ovarian Research (2022) 15:137 the main components of the ovary containing oocytes and granulosa cells [1]. Females are born with a limited number of oocytes (around 1-2 million), which are placed in primordial follicles [2]. Menopause begins when the woman's follicular reserve is depleted, and she is no longer able to conceive naturally [3]. On the other hand, studies reported that both pre-and post-menopausal ovaries contain pluripotent/multipotent stem cells that may differentiate into multiple cell lineages [4][5][6]. Stimpfel et al. [4] investigated the adipogenic, osteogenic, neural, and pancreatic differentiation ability of stem cells derived from pre-and post-menopausal ovaries and demonstrated the high plasticity of the stem cells isolated from these ovaries. Somatic stem cells are a subgroup of normal tissues with self-renewal ability and the potential to create lineage-committed daughter cells, which are important for tissue regeneration and repair [7]. On the other hand, the culture of stem cells in different media (for instance, supplemented media with fetal calf serum, human serum, or platelet lysate) could affect their proliferation rate, function, and phenotype [8].
Fan et al. [9] and Wagner et al. [10] provided a map of isolated cells from reproductive-age ovaries and identified six groups of cells, including endothelial, immune, granulosa, smooth muscle, theca, and stroma cells. In tissue engineering, cells play a critical role in the constructed tissue. For instance, endothelial cells could accelerate vascularization, stem cells could improve regeneration, or theca cells are necessary for follicle development [1,[11][12][13]. Therefore, it is essential to identify various cell types in a tissue and their fate after culturing in different media to optimize the design of an engineered tissue. In this study, we characterize isolated cells from menopausal ovarian cortex before and after in vitro culture using four different supplements: fetal bovine serum (FBS), human serum albumin (HSA), platelet lysate (PL), and KnockOut serum replacement (KO).

Cell culture
Each cell culture medium consisted of Dulbecco's modified Eagle's medium F-12 nutrient mixture (DMEM/F12; 21,041-025; Gibco), 1% antibiotic and antimycotic (Anti-Anti; A5955; Gibco), and 10% FBS, HSA, PL, or KO. The cells were in vitro cultured at 37 °C in a humidified incubator with 5% CO 2 and the medium was replaced every other day. After 16 days, the cells were detached using Accutase (A6964; Sigma-Aldrich) to perform multiparametric flow cytometry analysis. Furthermore, the light microscopy analysis on cell morphology and proliferation on days 5, 7, 9, and 15 was performed.

Flow cytometry
Human ovarian isolated and cultured cells were processed for flow cytometry according to the Feisst et al. [18] procedure to determine the cell populations. Before staining, the frozen-thawed cells were washed and incubated in FBS culture medium at 37 °C, 5% CO 2 for 1 h. After washing with DPBS, frozen-thawed and cultured cells were stained with the following antibodies ( BioLegend), and CD105 (323,205; BioLegend) and darkincubated at room temperature for 10 min. At least 10,000 events were analyzed by BD FACS-Canto ™ II Clinical Flow Cytometry System (BD Biosciences, Belgium). FlowJo software (BD Biosciences, USA) was used for data analysis.

Statistical analysis
One-way ANOVA was used to examine the data and statistical significance was determined by P values less than 0.05. The quantitative data were presented as mean ± SD and a single sample standard deviation is represented by the error bars in graphs.

Flow cytometry
In order to investigate the identity of ovarian stromal cells after isolation (D0) and culture in different media supplemented by HSA, FBS, PL, and KO, the cells were stained with 8 cell markers. These supplements are different from each other and their composition can therefore trigger different cell behaviors. FBS is an undefined serum, and its composition varies from lot to lot and depends on the diet and environment of a pregnant female. Indeed, Zheng et al. [19] investigated the growth of adult retinal pigment epithelial cells in three different FBS batches and reported that the growth rate of cells was significantly higher in one of the batches compared to others. While it is not possible to establish the exact composition of FBS, some of its contents have already been described in the literature, such as different types of hormones (folliclestimulating hormone, glucagon, insulin, and thyroid hormones), growth factors and cytokines (basic fibroblast growth factor, endothelial cell growth factor, epidermal growth factor, and fibroblast growth factor) and other proteins (albumin, fibronectin, laminin, and transferrin) [20]. To reduce uncontrolled cell differentiation and avoid endotoxin and large variability observed in the FBS, this supplement has been replaced by other additives, such as serum-free medium, animal-free components, such as PL, and more defined serum replacement such as KO [21][22][23].
Studies have shown that undesired differentiation of cells cultured in a medium supplemented with KO is lower than FBS [24,25]. KO is composed of well-defined growth factors, amino acids, vitamins, antioxidants, trace elements, and proteins, including transferrin (iron-saturated), insulin, and lipid-rich albumin (AlbuMAX) [26]. Nevertheless, it is important to bear in mind that the exact composition of KO is not provided by its supplier. Moreover, KO is not the completely free of animal components because of the presence of AlbuMAX [27] and embryo extracts [28]. Albumin is an important protein with antioxidant function as well as a role in binding and carrying biological important factors for cells. Moreover, surfaces coated with albumin cause non-adhesive surfaces, preventing cell attachment [29,30]. Although HSA has been employed in cell culture, such as human endothelial cells, in which HSA acted as an apoptotic inhibitor [31], studies from human islet and embryo culture in FBS or FBS + HSA indicated superior results than HSA alone regarding oxygen consumption rate per DNA content or blastocyst implantation rate [32,33]. A more recent alternative to FBS is PL, an animal-free product, produced from human platelets, which are lysed several freezing and thawing cycles. PL contains HSA as a major protein component as well as different types of growth factors, such as fibroblast growth factor (FGF), endothelial growth factor (EGF), platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), and epidermal growth factor (EGF) originated from human plasma and platelet components [34,35]. However, PL is also a donor-related product, and a decrease in the concentration of PDGF, TGF, FGF, or IGF with an increase in donor age has been shown [36]. Therefore, in order to decrease batch variation, PL is generally produced from multiple donors. Based on such differences in these supplement (HSA, FBS, PL, and KO) compositions, it is indeed expected that they can regulate cell behavior differently. For instance, mesenchymal stem cells proliferate faster in medium supplemented with PL than with FBS [37].
In vitro culture in medium supplemented with KO medium yielded a significantly higher proportion of CD326+ cells (epithelial-like cells) compared to D0 (25.19 ± 22.7% vs. 0.72 ± 0.38%; p < 0.05) (Fig. 1). On the other hand, other medium additives had no significant differences in the CD326+ population.

Light microscopy analysis of cells in different media
Images of the cells cultured in different media were taken during in vitro culture on days 5, 7, 9, and 15 by using a light microscope (Fig. 9). The microscopy analysis of cells indicated that while the seeding density was similar in all groups at day 0, the population of the cells revealed slight differences among the groups after 3 days of in vitro culture. Cells in PL showed the highest confluence, and those in KO, the lowest. However, when comparing cell growth from day 3 to day 5, 9, and 15, both PL and FBS groups displayed a marked increase in cell number over time with 100% confluence at day 15, while the cell number in the KO and HSA groups showed no notable increase in cells from day 5 to day 15. These differences between the various conditions indicate the potential presence of different cell types when culturing stromal cells by using various supplements.
The cells displayed high adhesion to the flask surface when media supplemented with FBS and PL, and low adhesion when media supplemented with HSA and KO. Cells within all four groups grew in a monolayer and had an elongated, bi-or multipolar cell type, similar to fibroblasts. Within this monolayer, various sizes of cells can be seen, especially in the PL, FBS, and KOsupplemented media. A difference in the HSA group relative to the other groups is the round cells, similar to lymphoblasts. These dissimilarities within the same condition indicate the possibility of more than one cell type in each supplemented medium (Fig. 9).

Conclusion
In conclusion, our findings show that isolated cells from the ovarian cortex have distinct characterization when cultured in media supplemented with HSA, PL, KO, or FBS. Furthermore, the cells were identified by different Fig. 8 Representative dot plot for CD90, CD73, CD105, and CD146 expressed cells of CD31 + CD34-gate in different media (HSA, PL, KO, and FBS); n ≥ 3, mean ± SD; the asterisks demonstrate significant differences of CD90 + CD73+, CD90 + PDPN+, and CD105 + CD146+ with other gates; **p < 0.01; ***p < 0.001 phenotypes when they were cultured in media using one of these four additives. Most cells in KO and FBS expressed CD31-CD34-CD73 + CD90+, indicating the possibility of cells being fibroblasts, mesenchymal stem cells, or pericytes in these media. Also, the cells in KO medium indicated a significant increase in cells expressed CD326+ (epithelial cells). PL and HSA groups contained more CD31 + CD34 + CD90 + PDPN+, lymphatic cells compared to the other group as well as PL demonstrated a significant increase in the CD31 + CD34-, endothelial cells compared to day 0. While this initial study offers a novel strategy to obtain different cell populations for the bioengineered ovary, further studies, such as single-cell RNA-sequencing, would be instrumental in precisely identifying the different cell populations before and after in vitro culture. Identifying cells before their use for regenerative medicine is crucial as different cell types secret different growth factors and cytokines that can direct the fate of engineered tissue and the formation of extracellular matrix.